Recombinant approaches have been used in attempts to develop vaccines against diseases for which no vaccine currently exists, or for which conventional vaccine approaches are less desirable. For example, since the human immunodeficiency virus (HIV) was first identified as the etiologic agent of Acquired Immunodeficiency Disease Syndrome (AIDS), (Barre-Sinoussi et al. Science 220:868 (1983); Levey et al., Science 225:840 (1984); Gallo et al., Science 224:500 (1984)), considerable effort has been directed towards the development of a safe and effective vaccine.
The human immunodeficiency viruses, HIV-1 and HIV-2, are members of the lentivirus subclass of retroviruses. Gonda et al., Science 227:173 (1985); Sonigo et al., Cell 42:369 (1985). The virus particles contain an inner core comprised of capsid proteins (encoded by the viral gag gene) that encase the viral RNA genome. Rabson & Martin, Cell 40:477 (1985). The central core is surrounded by a lipid envelope that contains the viral-encoded envelope glycoproteins. Virus-encoded enzymes required for replication, such as the reverse transcriptase and integrase (encoded by the pol gene), are also incorporated into the virus particle.
Simian immunodeficiency virus (SIV) is a virus closely related to HIV. Several isolates of SIV have been cloned and sequenced. The results reveal 40-50% overall identity in the predicted amino acid sequences when compared to HIV-1 and about 75% when compared to HIV-2. Experimental inoculation of this virus into macaque monkeys has consistently resulted in long-term persistent infection, with most inoculated animals dying of a disease similar to AIDS in humans. A number of researchers have reported successful vaccination of macaques with whole inactivated SIV with protection against subsequent challenge with lethal doses of SIV (Desrosiers et al., Proc. Natl. Acad. Sci. USA, 86:6353 (1989); Murphey-Corb et al., Science, 246:1293 (1989)).
There are obvious difficulties with the use of whole virus for an HIV vaccine. The fear that an attenuated virus could revert to virulence, and the danger of incomplete inactivation of killed virus preparations, together with the reluctance to introduce the HIV genome into seronegative individuals have argued against the uses of live attenuated or killed HIV vaccines for the prevention of infection.
Advances in recombinant DNA technology may make it possible to use heterologous expression systems for the synthesis not only of individual antigens, but also of defective, nonself-propagating, virus-like particles. It has been demonstrated that capsid proteins of certain viruses can assemble into particles morphologically and immunologically similar to the corresponding virus. For example, the P1 precursor of several picornaviruses synthesized in vitro can be processed into individual capsid proteins which then assemble into immunoreactive virion-like particles. Nicklin et al., Biotechnology 4:33 (1986; Palmenberg et al., J. Virol. 32:770 (1979); Shih et al., Proc. Natl. Acad. Sci. USA 75:5807 (1978); Hanecak et al., Proc. Natl. Acad. Sci. USA 79:3973 (1982); Grubman et al., J. Virol. 56:120 (1985). Self-assembly of capsid proteins expressed in vivo in several recombinant expression systems has also been reported. For example, when human hepatitis B surface antigen is expressed in yeast cells, the polypeptide assembles into particles similar in appearance to those isolated from human plasma (Valenzuela et al., Nature 298:347 (1982)); these particles stimulate anti-hepatitis B antibody production in several species and can protect chimpanzees from virus challenge. McAleer et al., Nature 307:178 (1984).
In another example, it was shown that coexpression of canine parvovirus (CPV) capsid proteins VP1 and VP2 in murine cells transformed with a bovine papilloma virus/CPV recombinant plasmid resulted in the formation of self-assembling virus-like particles that resembled, biochemically and immunologically, authentic CPV virions (Mazzara et al., 1986, in Modern Approaches to Vaccines, Cold Spring Harbor Laboratory, N.Y.; R. M. Chanock and R. A. Lerner, eds. pp. 419-424; Mazzara et al., U.S. patent application No. 905,299, filed Sep. 8, 1986). When used to vaccinate susceptible dogs, these empty capsids elicited immune responses capable of protecting against CPV challenge. It has also been shown that the HIV-1 or SIV p55gag precursor polypeptide expressed in insect cells using the baculovirus expression system results in the formation of immature, retroviral-like particles that are secreted into the cell culture medium of infected cells. Gheysen et al., Cell 59:103 (1989); Delchambre et al., The EMBO J. 8:2653-2660 (1989).
In mammalian cells, HIV-like particles that contained core polypeptides as well as reverse transcriptase were produced after transient expression of the HIV gag-pol genes using an SV40 late replacement vector (Smith et al., J. Virol. 64:2653-2659 (1990). Mammalian cells infected with recombinant vaccinia virus containing the HIV gag-pol genes have also been shown to produce defective,.HIV-like particles (Karacostas et al., Proc. Natl. Acad. Sci. USA, 86:8964 (1989)).
Recombinant fowlpox virus (FPV) has also been used as a vector for the expression of foreign genes. Fowlpox virus is an avipox virus distantly related to vaccinia virus, an orthopox virus. Recombinant fowlpox viruses containing foreign DNA within a region of the viral genome which is nonessential for growth in tissue culture have been described by Boyle et al. International Patent Application PCT/AU87/00323, Boyle and Coupar (1988) Virus Res. 10:343. Vaccinia virus promoters are used to express the DNA in FPV.
Several other groups have published the construction of FPV recombinantso Noboru et al., (EPO 284,416, filed Mar. 25, 1988) disclose a number of genomic insertion sites which are nonessential for FPV growth in tissue culture, using the E. coli lacZ gene under the control of a vaccinia promoter. Paoletti (PCT/US88/02816, filed Aug. 24, 1988; Taylor et al., (1988) Vaccine 6: 497-503, 504-508) describes vectors for producing FPV recombinants using various vaccinia promoters for the expression of genes encoding foreign antigens, including the rabies G protein, turkey influenza hemagglutinin and avian bronchitis virus spike protein. Drillien and Spehner (EPO 314,569, filed Oct. 26, 1988) disclose the construction of FPV recombinants containing a gene encoding the measles F protein under the control of a vaccinia promoter.
Productive fowlpox infection is restricted in vivo to avian species and in vitro to cells derived from avian species. Fowlpox virus does cause cytotoxic effect in mammalian cells (Burnett and Frothingham, Archiv fur die gesamte Virusforschung, 24:137 (1968)). The cytotoxic effect was not visible until three days post-infection with fowlpox virus and maximal effect was not observed until seven to nine days post infection. Pretreatment of fowlpox virus with ultraviolet light reduced the cytotoxic effect. These observations, i.e., the delayed onset of cytotoxic effect and the inhibitory effect of ultraviolet light, indicate that the cytotoxic effect of fowlpox virus in mammalian cells is not due to a previously synthesized toxic factor carried in with the infecting fowlpox virion, but is more likely due to de novo production of fowlpox viral material in the infected mammalian cell. Thus, these studies imply that a certain level of fowlpox viral gene expression may be occurring in mammalian cells, without production of infectious material. More recently, Paoletti (PCT/US88/02816, filed Aug. 24, 1988) and Taylor et al., (Vaccine, 6:497-503 (1988) confirmed that fowlpox viral gene expression occurs in infected non-arian cells. Native fowlpox gene expression was not investigated, but expression of foreign genes under the control of a vaccinia promoter was demonstrated in monkey or human cells infected with recombinant fowlpox virus containing these sequences. The techniques used to detect such foreign protein synthesis (e.g., radioimmunoprecipitation) are very sensitive and can therefore detect very low levels of gene expression. It is thus not clear whether quantitatively useful amounts of protein were made, for example, to be able to use fowlpox infection of mammalian cells for protein production. Sufficient protein was made to elicit a protective immune response against several pathogens. However, in many cases, especially in the case of rabies G protein, very low levels of antigen are needed to elicit a protective immune response.